In the present disclosure, a substance having a molecular weight of less than 500 Daltons is denoted as a compound and a substance having a molecular weight of 500 Daltons or more is denoted as a polymer.
Covalent modification of the surfaces of the micro/nano materials with hydrophilic compounds/polymers, such as polyethylene glycol (PEG), compounds/polymers of zwitterion, or hydrophilic natural biopolymers (e.g., heparin and albumin, etc.) can reduce their non-specific adsorption to enhance their applications. Natural biopolymers are susceptible to non-specific adsorption of other biomolecules and still have strong non-specific adsorption of hydrophobic small molecules. For covalent modification of hydrophobic micro/nano materials, PEG and zwitterionic compounds are more attractive because their modification layers significantly reduce non-specific adsorption while possess stable and inert chemical structures. When the PEG chain is long enough, the modification effect is significant, otherwise a very high degree of modification is needed; the flexibility of the PEG chain itself limits the degree of modification; when the PEG density on the surface of the modified micro/nano materials is too large, the PEG chain still has non-specific adsorption of both proteins and hydrophobic small molecules. Therefore, the actual effect of modification with PEG alone is insufficient, making the covalent modification with zwitterions the sole promising way.
Zwitterions, also known as amphoteric ions, refer to functional groups/compounds with equal amounts of positive and negative charges. The zwitterions modification agent coated on the surface of the micro/nano material can significantly reduce non-specific adsorption. In addition, a mixture of a large number of intermolecular ion pairs also has a strong hydration ability and a weak non-specific adsorption. However, the accessible zwitterion modification agents have small sizes, leading to small modified areas and weak modification effects. Therefore, it is necessary to optimize the scheme of modification with zwitterions.
The design of the modification scheme firstly needs a selected covalent chemical reaction. Under mild conditions, a carboxyl group is easily activated into an active ester at a high yield, and then reacts with a primary or secondary aliphatic amine to form an amide bond at a high speed and high yield. The formation of amide bonds is thus a suitable covalent modification reaction, providing a high density of carboxyl groups on the surface of the micro/nano material for the conversion of such carboxyl into active esters which are reacted with a modification agent having the primary and/or secondary aliphatic amines.
The design of the modification scheme also needs the selection of the structure of the modification agent and the modification reaction conditions. A zwitterionic compound containing one or more primary and/or secondary aliphatic amines as a modification agent is usually insoluble in organic solvents but is easily soluble in an aqueous solution. This modification agent needs a modification reaction in an alkaline aqueous solution with an active ester on the surface of the micro/nano material. Otherwise, the amino reactivity is low and the degree of modification is insufficient, but the active ester is hydrolyzed in an alkaline aqueous solution so rapidly that the degree of modification still cannot be guaranteed. The active ester is stable under weakly acidic conditions, but the amine ion is ionized under such conditions and reacts slowly with the active ester, leading to uncertain degree of modification. The active ester reacts rapidly with the primary and secondary aliphatic amines in an inert organic solvent with high yields even at room temperature, but the zwitterionic compound is difficult to be dissolved in such an inert organic solvent. The solubility of nonionic compounds in inert organic solvents is generally high. A nonionic compound containing primary and/or secondary aliphatic amines and one or more functional groups which produce the amphoteric ion pairs by a simple reaction (i.e., pro-zwitterion groups), is designed as a modification agent. In an inert organic solvent, the modification agent forms amides with the active esters on the surface of the micro/nano material, and then the pro-zwitterion groups in situ are derived to produce zwitterions with high yields, giving a covalent modification layer of abundant zwitterions on the surface of the modified micro/nano material. This approach may be the sole strategy to generate a covalent modification layer of abundant zwitterions on the surface of the modified micro/nano material for negligible nonspecific adsorption of common substances.
1,3-propyl sultone can react with non-amide primary, secondary and tertiary aliphatic amines substituted by small alkyl groups to produce covalently bonded sulfonic acid. The number of sulfonic acid groups that are linked to a primary or secondary aliphatic amine is uncertain during covalent modification of a micro/nano material, yielding a charged surface that easily causes non-specific electrostatic adsorption of substances charged. Namely, the modification reaction with the micro/nano material is heterogeneous, and thus it is impossible to form a uniform zwitterion-modified neutral surface by controlling the amount of 1,3-propyl sultone. However, a tertiary amine bearing three small alkyl substituents can only be linked to one sulfonic acid and produce an amphoteric ion pair, thus being a suitable pro-zwitterion group. Therefore, a modification agent containing such tertiary amines is used to react with active esters on the surface of the micro/nano material in an inert organic solvent, and then 1,3-propyl sultone is used to convert such tertiary amines in situ to amphoteric ion pairs as completely as possible. This may be the only practical way to greatly reduce the non-specific adsorption by covalent modification of micro/nano materials with amphoteric ion pairs.
A hydrophilic modification agent bearing pro-zwitterion groups can significantly reduce the non-specific adsorption of the modified micro/nano materials only when the modification agent is coated on the surface of the micro/nano material at sufficient density, which requires a modification agent having a bulky size and a modification degree as high as possible. However, the steric hindrance of a bulky modification agent reduces both its reactivity with the active esters on the surface of the micro/nano material and the degree of modification. Inserting a linear linking arm between the modification agent and the active esters on the surface of the micro/nano material facilitates improving the coverage of the surface of the micro/nano material by the modification agent, while reducing the steric hindrance of the modification reaction. Covalent modification in an alternate mode with a small-sized modification agent bearing pro-zwitterion group(s) and then with a short PEG arm helps to enable the degree of modification in each step of the modification reactions. A multilayer covalent modification with a reasonably-sized modification agent bearing pro-zwitterion group(s) gradually increases the coverage of the surface of the micro/nano material by the modification agent, which is a necessary way to avoid the steric hindrance effect of the bulky modification agent itself and to enable the degree of modification. Inserting the linear linking arm directly or indirectly between the modification agent and the active esters on the micro/nano material surface facilitates improving both the coverage of the surface and the degree of modification. The modification reaction on the surface of the micro/nano material is heterogeneous with low repeatability, which requires a minimized number of modification reactions for a uniform product after modification. The use of a hydrophilic modification agent bearing pro-zwitterion group with a short linear hydrophilic linking arm to the primary and/or secondary amines (directly inserted), or the extension of the active esters on the surface obtained after each modification step to provide a linear linking arm indirectly, improves the coverage of the surface by the modification agent.
In order to guarantee the degrees of re-modification after each modification reaction for the formation of multiple modification layers, the pro-zwitterion modification agent used in each modification reaction must have sufficient numbers of carboxyl groups for conversion into active esters followed by covalent modification, but the potential electrostatic repulsions between these carboxyl groups may reduce both the reactivity of the modification agent itself with the active esters on the surface of the micro/nano material and the modification degree. The use of a neutral group which is a pro-carboxyl group, which can be converted into a carboxyl group through a simple reaction, can prevent the electrostatic repulsions between carboxyl groups from hindering the modification reaction, so as to guarantee the degree of modification. The use of monomers containing a large amount of neutral pro-carboxyl groups to prepare the micro/nano material by polymerization also facilitates deriving more active esters on the surface, which is an important strategy for making the micro/nano materials suitable for modification according to the present disclosure. Obviously, the use of a modification agent containing the pro-carboxyl group, the pro-zwitterion group, the primary/secondary aliphatic amine as the reactive functionality(ies) and the hydrophilic flexible linking arm(s) to such reactive functionality(ies), together with the use of monomer(s) containing neutral pro-carboxyl groups for polymerization to make a micro/nano material, is a necessary and comprehensively-optimized scheme for covalent modification.
In applications, it is desirable to retain the activity of the biomolecule immobilized on the surface of the micro/nano material through site-specific immobilization. In immunoassays, antibodies are often immobilized on the surface of micro/nano materials or form covalent adducts with micro/nano materials. Natural antibodies have several pairs of non-essential disulfide bonds away from the antigen binding site. The signal molecule/material having no sulfhydryl group and no disulfide bond on the surface can be conjugated to antibodies through selective covalent modification of the antibody disulfide bonds, which is advantageous for retaining the activities of both parts in the adducts and increasing the yields of the adducts. Hence, the present disclosure also utilizes a moiety free of sulfhydryl groups and disulfide bonds but selective for protein disulfide bonds, for the selective covalent labeling of an antibody by a protein having no sulfhydryl groups and no disulfide bonds on the surface, and the asymmetric cross-linking of a polymer free of sulfhydryl groups and disulfide bonds or such a small biochemical with the antibody, to give a higher activity in the resulting adduct.
Therefore, the core idea of the present disclosure is a systematically-optimized comprehensive solution for obtaining micro/nano materials bearing surface hydrophilicity and immobilized biomolecules with as high activities as possible, and the technical points thereof include: (1) optimization of the preparation of micro/nano materials to be modified: the micro/nano materials are prepared through the polymerization of special organic monomers where more aliphatic carboxyl or/and pro-carboxyl groups are formed on the surface of the resulting micro/nano material and converted into active esters at higher yields; (2) optimization of the modification agent structure: a hydrophilic modification agent of appropriate size having primary and/or secondary aliphatic amine, pro-zwitterion group(s) or/and carboxyl and/or pro-carboxyl groups(s), and a flexible/linear hydrophilic linking arm is employed as the modification agent; (3) optimization of the modification reaction: the optimized modification agent is used to form amide bonds in a high yield with the active esters on the surface of the micro/nano material in an inert organic solvent, thereby improving the degree of modification on the surface of the material; (4) optimization of the coverage of material surface with the modification agent: the coverage on the material surface with the modification agent is increased by multilayer covalent modifications obtained through repeated covalent modification to prevent the steric hindrance of a bulky hydrophilic modification agent from reducing the degree of modification and coverage; (5) the functional groups are provided with the flexible linking arm during the multilayer covalent modification process with the hydrophilic modification agent, and the specific functional groups on the surface of the covalently modified micro/nano material improves the retention of biological activity of immobilized macromolecules. The systematic optimization and integration of the various strategies effectively reduce the non-specific adsorption of common substances on the modified material and increase the activities of the immobilized macromolecules.